This application is filed as a 371 application based on PCT/EP99/03032 filed Apr. 28, 1999 which claims priority to UK 9810016.7 filed May 8, 1998.
This invention relates to coumarin derivatives, processes for preparing them and their use as enzyme substrates.
The majority of metabolism based drug interactions are a result of inhibition of cytochrome P450 enzymes. Drug interactions involving individual P450 enzymes can be predicted using in vitro methods. Typical in vitro P450 enzyme assays involve incubation of an appropriate substrate with a source of enzyme. Traditionally, time consuming chromatographic methods have been used for metabolite detection in these incubations. More recently the availability of fluorimetric plate readers has facilitated the higher throughput of enzyme assays in general. Adapting P450 assays to fluorescent plate reader technology requires the identification of substrates with appropriate fluorescent products for individual enzymes. Among the xenobiotic-metabolising cytochromes P450, CPY2D6 is one of those commonly responsible for the metabolism of drugs.
3-Cyano-7-ethoxycoumarin has been described for CYP2D6 inhibition screening (Crespi et al, Anal. Biochem., 1997, 248, 188-190). Other CYP2D6 substrates have also been described (Koymans et al, Chem. Res. Toxicol., 1992, 5, 211-219), however these do not give fluorescent products suitable for high throughput screening.
Certain compounds have now been identified which are improved substrates for CYP2D6 and which are of use for configuring high throughput inhibition screening assays.
As a brief description of the drawings: FIG. 1 shows inhibition of 7-methoxy-4-(methylarninomcthyl)coumarin metabolism with quinidine.
According to the present invention there is provided an assay for testing for inhibitors of the enzyme CYP2D6 which comprises contacting the enzyme and a compound of formula (I): 
wherein R1 and R2 independently represent hydrogen or C1-2alkyl and R3 is C1-2alkyl, with a test compound and measuring inhibition of O-dealkylation of the compound of formula (I) by the enzyme.
Preferably R1 is hydrogen and R2 is methyl
R3 is preferably methyl.
Generally the rate of O-dealkylation of the compound of formula (I) in the absence of test compound will be known, as will the extent of O-dealkylation at given time points. The assay may test for inhibition of O-dealkylation continuously or at specified time points.
O-dealkylation of the compound of formula (I) by CYP2D6 gives a readily quantifiable fluorescent product of formula (II): 
wherein R1 and R2 are as defined for formula (I), which can be scanned with suitable excitation and emission wavelengths, for example an excitation wavelength of 409 nm and an emission wavelength of 485 nm.
The assay may be carried out either in solution or utilising a solid support. When the assay is carried out in solution suitable solvents include methanol, acetonitrile and DMSO.
The test compound may be pre-incubated with enzyme prior to the addition of the substrate, or alternatively the substrate may be added simultaneously. Final concentrations of enzyme and substrate are calculated so as to achieve a suitable rate of processing for carrying out the assay. If desired, the reaction may be stopped, for example by addition of acid or solvent. The product may be analysed using any conventional system of fluorescence detection, for example a multi-well plate/fluorescent plate reader.
Certain of the compounds of formula (I) are novel, thus according to a further aspect of the invention there is provided a compound of formula (I), as defined above, wherein at least one of R1 and R2 represents methyl.
Certain of the compounds of formula (II) are novel, thus according to a further aspect of the invention there is provided a compound of formula (II), as defined above, wherein R1 is methyl and R2 is hydrogen or ethyl.
The compounds of formula (I) may be prepared by conventional methods, for example as shown in Scheme 1: 
Thus according to a further aspect of the invention there is provided a process for the production of a compound of formula (I) which comprises reaction of a compound of formula (III): 
wherein L is a leaving group e.g. Br, and R3 is C1-2alkyl, with a compound of formula (IV):
NHR1R2xe2x80x83xe2x80x83(IV)
wherein R1 and R2 are as defined for formula (I) provided that at least one of R1 and R2 represents methyl.
The reaction is preferably performed in the presence of a base, e.g. potassium carbonate.
Since the inhibition of cytochrome P450 enzymes is often the mechanism for drug/drug interactions, the assay according to the invention is particularly useful for identifying compounds which may give rise to adverse drug/drug interactions. The assay can therefore be used in combination with the chemical modification of test compounds to increase a test compound""s potential for use as a pharmaceutical.
Thus according to further aspects of the invention there are provided a method for reducing the CYP2D6 enzyme inhibitory activity of a compound, comprising the steps of identifying the compound as an inhibitor of CYP2D6 in the assay described above; and thereafter producing a chemically modified version of the test compound in which the functionality suspected to be responsible for CYP2D6 inhibition is eliminated or changed; and novel compounds produced according to this method.
The chemical modification of test compounds according to this method can be performed using techniques well known to those skilled in the art.
The novel compounds produced according to this aspect of the invention may find application as pharmaceuticals. The fact that a compound produced according to this method will be readily identifiable as novel by performing routine literature and database searches. The pharmaceutical activity of such compounds can be readily ascertained using conventional biological screening methods known to those skilled in the art.